Method of treating glaucoma comprising administering a composition comprising human BMP7

ABSTRACT

The present invention provides methods and kits for diagnosing and treating glaucoma.

The present application is a divisional of U.S. patent application Ser.No. 12/106,653 filed Apr. 21, 2008 (now allowed), which claims priorityto U.S. patent application Ser. No. 10/286,152 filed Oct. 31, 2002 (nowU.S. Pat. No. 7,405,192), which claims benefit to ProvisionalApplication Ser. No. 60/334,852 filed Oct. 31, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention discloses methods and reagents for diagnosing andtreating glaucoma and related disorders.

2. Description of the Related Art

“Glaucomas” are a group of debilitating eye diseases that are theleading cause of irreversible blindness in the United States and otherdeveloped nations. Primary Open Angle Glaucoma (“POAG”), the most commonform of glaucoma, is characterized by the degeneration of the trabecularmeshwork, leading to obstruction of the normal ability of aqueous humorto leave the eye without closure of the space (e.g., the “angle”)between the iris and cornea (Vaughan, D. et al., (1992)). Acharacteristic of such obstruction in this disease is an increasedintraocular pressure (“IOP”), resulting in progressive visual loss andblindness if not treated appropriately and in a timely fashion. Thedisease is estimated to affect between 0.4% and 3.3% of all adults over40 years old (Leske, M. C. et al. (1986); Bengtsson, B. (1989); Strong,N. P. (1992)). Moreover, the prevalence of the disease rises with age toover 6% of those 75 years or older (Strong, N. P., (1992)).

Because increased IOP is a readily measurable characteristic ofglaucoma, the diagnosis of the disease is largely screened for bymeasuring intraocular pressure (tonometry) (Strong, N. P. (1992); Greve,M. et al. (1993)). Unfortunately, because glaucomatous and normalpressure ranges overlap, such methods are of limited value unlessmultiple readings are obtained (Hitchings, R. A., (1993); Tuck, M. W. etal. (1993); Vaughan, D. et al., (1992); Vernon, S. A., (1993)). For thisreason, additional methods, such as direct examination of the optic diskand determination of the extent of a patient's visual field loss areoften conducted to improve the accuracy of diagnosis (Greve, M. et al.,(1993)).

Glaucoma affects three separate tissues in the eye. The elevated IOPassociated with POAG is due to morphological and biochemical changes inthe trabecular meshwork (TM), a tissue located at the angle between thecornea and iris. Most of the nutritive aqueous humor exits the anteriorsegment of the eye through the TM. The progressive loss of TM cells andthe build-up of extracellular debris in the TM of glaucomatous eyesleads to increased resistance to aqueous outflow (Lutjen-Drecoll andRohen 1996; Rohen 1983; Rohen et al. 1993; Grierson and Calthorpe 1988),thereby raising IOP. Elevated IOP, as well as other factors such asischemia, cause degenerative changes in the optic nerve head (ONH)leading to progressive “cupping” of the ONH (Varma and Minckler 1996;Hernandez and Gong 1996; Hernandez et al. 1990; Hernandez and Pena 1997;Morrison al. 1990) and loss of retinal ganglion cells (Quigley et al.2000; Quigley 1999; Quigley et al. 1995; Kerrigan et al. 1997) andaxons. The detailed molecular mechanisms responsible for glaucomatousdamage to the TM, ONH, and the retinal ganglion cells are unknown.

Current glaucoma therapy is directed to lowering IOP, a major riskfactor for the development and progression of glaucoma. These therapieslower IOP, but they do not directly address the pathogenic mechanisms,and the disease continues to progress. At least half of patients withglaucoma are undiagnosed, and by the time patients are diagnosed withglaucoma, they have already lost approximately 40% of their retinalganglion cells. Therefore, methods for earlier detection and diagnosisof glaucoma are needed.

In view of the importance of glaucoma, and the at least partialinadequacies of prior methods of diagnosis, it would be desirable tohave an improved, more accurate method for diagnosing glaucoma in itsearly stages. In addition, it would be desirable to have new therapeuticagents that address glaucomatous pathogenic mechanisms.

SUMMARY OF THE INVENTION

The present invention overcomes these and other drawbacks of the priorart by providing methods and kits for the early diagnosis of glaucoma,for treating glaucoma, and for the identification of compounds useful inthe treatment of glaucoma.

In certain specific embodiments, the invention provides a method fordiagnosing glaucoma in a sample obtained from a cell or bodily fluid bydetecting altered expression of a bone morphogenic protein family membergene. The method generally includes the steps of:

-   -   a) obtaining a tissue or fluid sample from a patient suspected        of having glaucoma;    -   b) extracting DNA from said sample;    -   c) obtaining a plurality of PCR primers, wherein said primers        each comprise a sequence consisting of from 18 to 1547        contiguous nucleotides from SEQ ID NO:1, SEQ ID NO:3, SEQ ID        NO:5, SEQ ID NO:7, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:41,        SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:53;    -   d) amplifying regions of the extracted DNA using said primers to        obtain a PCR product;    -   e) resolving the PCR product; and    -   f) identifying differences between the sequence of the PCR        product and the normal gene sequence;    -   where a difference between the amplified sequence and the normal        gene sequence is diagnostic of glaucoma.

In general, the methods of the invention may include obtaining a samplefrom an individual and extracting DNA from said sample. Select PCRprimers for specific members of the BMP gene family are then used toamplify relevant regions of the extracted gene to obtain a PCR product.The PCR product is resolved by a technique that effectively identifiesDNA sequence differences between the normal and mutated form of thespecific BMP family gene being evaluated (the extracted DNA). Identifieddifferences between the sequences are indicative of glaucoma.

The tissue or fluid sample for use in the methods of the invention maybe blood or buccal cells.

Typically, the primer sequences will have a length of between about 10,15 or 18 nucleotides to about 20, or to about 30 nucleotides. Longersequences, e.g., 40, 50, 80, 90, 95, 100, even up to full length, areeven more preferred for certain embodiments. Lengths of oligonucleotidesof at least about 18 to 20 nucleotides are well accepted by those ofskill in the art as sufficient to allow sufficiently specifichybridization so as to be useful as a molecular probe, as described byLathe (1985), which reference is specifically incorporated herein byreference for this purpose. Preferably, the nucleotide sequence willconsist of from 20 to 100 contiguous nucleotides of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:53. It isalso contemplated that the primer sequences may consist of sequences ofat least 10, 15 or 18 contiguous nucleotides from the sequences of BMPreceptor genes and from BMP-associated proteins, the sequences of whichare known.

Nucleic acid molecules having stretches of 10, 18, 20, 30, 50, 60, 65 oreven up to and including 100 nucleotides or so, complementary to any oneof SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:37,SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, orSEQ ID NO:53, have utility as hybridization probes. Primers or probeshaving a nucleotide length of about 18 nucleotides are recognized bythose of skill in the art to provide highly specific hybridization to atarget sequence. The total size of the fragment, as well as the size ofthe complementary stretches, will ultimately depend on the intended useof application of the particular nucleic acid segment. Smaller fragmentswill generally find use in hybridization embodiments, wherein the lengthof the complementary region may be varied, such as between about 10, 18,20 or 30 and about 50, 60, 70, 80, 80 or 100 nucleotides, or even fulllength according to the complementary sequences one wishes to detect.

In specifically preferred embodiments, the primers will consist ofcontiguous sequences from SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, or SEQ ID NO:53. In other preferred embodiments,the primers will consist of contiguous sequences from BMP receptor genes(disclosed in ten Dijke et al. 1993; Astrom et al. 1999; Nohno et al.1995, all incorporated herein by reference) or from BMP-associatedgenes, such as chordin (NCBI NM_(—)029130), gremlin (Murphy et al. 1999;McMahon et al. 2000), follistatin (NCBI NM_(—)003892) or bambi (NCBINM_(—)005791). Most preferably, the primers will consist of contiguoussequence from SEQ ID NO:3. In certain aspects, at least some of theprimers may further include a detectable label.

In other embodiments, the invention provides a method for treatingglaucoma by administering to a patient in need thereof a compositioncomprising a sequence consisting of at least one compound selected fromthe group consisting of a BMP2 agonist, a BMP4 agonist, a BMP5 agonist,a BMP7 agonist, a Smad 1/5 agonist, a chordin antagonist, a gremlinantagonist and a follistatin antagonist.

In additional aspects, the present invention provides a method foridentifying a therapeutic agent for the treatment of glaucoma.Therapeutic agents may be identified, for example, by:

-   -   a) obtaining a first composition comprising a population of        recombinant cells expressing BMP-2A, BMP4, BMP-5, or BMP7;    -   b) obtaining a candidate substance;    -   c) incubating said composition and said candidate substance;        testing said composition for its ability to turn on BMP-induced        Smad signaling pathways and/or BMP-regulated gene expression;        and identifying a candidate substance that inhibits, or        stimulates, these downstream effects of BMP.

Another aspect of the invention are diagnostic kits containing sequencesof the present invention and suitable reagents such as a detectablelabel linked to a protein, peptide or the antibody itself.Alternatively, the detectable label may be linked to a second sequencewhich selectively hybridizes to a sequence of the invention.

Related embodiments include therapeutic kits which includepharmaceutically-acceptable formulations of either the nucleic acidsequences or peptide or protein sequences disclosed herein. Such kitsare useful in the detection of altered expression of the BMP genes andproteins in clinical samples for the diagnosis of glaucoma.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1. Nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequenceof BMP2A.

FIG. 2. Nucleotide (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4) sequenceof BMP4.

FIG. 3. Nucleotide (SEQ ID NO: 5) and amino acid (SEQ ID NO: 6) sequenceof BMP5.

FIG. 4. Nucleotide (SEQ ID NO: 7) and amino acid (SEQ ID NO: 8) sequenceof BMP7.

FIG. 5. Bone morphogenic protein signaling pathway. Bone MorphogenicProtein (BMP) dimers bind to a membrane complex composed of BMPreceptors 1 and 2, which are serin/threonine kinases. The regulatorySmads (Smad1/Smad5) become phosphorylated and associate with a co-Smad(Smad 4). This resulting Smad complex enters the nucleus where itassociates with transcription factors (TF) and regulates geneexpression. BMP associated proteins act as BMP antagonists by bindingBMPs and preventing BMP interaction with BMP receptors.

FIG. 6. BMP expression in human TM cells and tissues. Ethidiumbromide-stained agarose gel of BMP PCR products from cDNA samplesgenerated from RT-PCR analysis of BMP expression in human TM cells(lanes 1-5) and tissues (lanes 6-7). L=base pair markers. C=PCR negativecontrol lane. β-actin was used as a positive RT-PCR internal control.

FIG. 7. BMP receptor expression in human TM cells and tissues. Ethidiumbromide-stained agarose gel of PCR products from cDNA samples generatedfrom RT-PCR analysis of BMP receptor expression in human TM cells (lanes1-5) and tissues (lanes 6-7). L=base pair markers. C=PCR negativecontrol lane. β-actin was used as a positive RT-PCR internal control.

FIG. 8. BMP expression in human ONH astrocytes, ONH tissues, and humanbrain astrocytes. Ethidium bromide-stained agarose gel of PCR productsfrom cDNA samples generated from RT-PCR analysis of BMP expression inhuman ONH astrocyte (lanes 1-5), ONH tissue (lane 6), and human brainastrocytes (lane 7). L=base pair markers. C=PCR negative control lane.β-actin was used as a positive RT-PCR internal control.

FIG. 9. BMP expression in human lamina cribrosa cell lines. Ethidiumbromide-stained agarose gel of PCR products from cDNA samples generatedfrom RT-PCR analysis of human lamina cribrosa cells (lanes 1-9). L=basepair markers. C=PCR negative control lane. β-actin was used as apositive RT-PCR internal control.

FIG. 10. BMP receptor expression in human ONH astrocytes, ONH tissues,and human brain astrocytes. Ethidium bromide-stained agarose gel of PCRproducts from cDNA samples generated from RT-PCR analysis of BMPreceptor expression in human optic nerve head astrocytes (ONA) (lanes1-5), ONH tissue (lane 6), and human brain astrocytes (lane 7). L=basepair markers. C=PCR negative control lane. β-actin was used as apositive RT-PCR control.

FIG. 11. BMP receptor expression in human lamina cribrosa cell lines.Ethidium bromide-stained agarose gel of PCR products from cDNA samplesgenerated from RT-PCR analysis of human lamina cribrosa cells (lanes1-9). L=base pair markers. C=PCR negative control lane. β-actin was usedas a positive RT-PCR control.

FIG. 12. Western immunoblot of BMP and BMP receptor expression incultured human TM cells, optic nerve head astrocytes (ONA), and laminacribrosa cells. Chemiluminescent detection of BMP proteins and BMPreceptors in human trabecular meshwork cells (lanes 1-2), ONH astrocytes(lanes 3-4), and lamina cribrosa cells (lanes 5-6). Protein sizeindicated in kDa.

FIG. 13. BMP associated protein mRNA expression in human TM cells.Ethidium bromide-stained agarose gel of PCR products from cDNA samplesgenerated from RT-PCR analysis of human TM cells (lanes 1-5). L=basepair markers. C=PCR negative control lane. β-actin was used as apositive RT-PCR internal control.

FIG. 14. BMP associated protein mRNA expression in human lamina cribrosacells and ONH astrocytes. Ethidium bromide-stained agarose gel of PCRproducts from cDNA samples generated from RT-PCR analysis of laminacribrosa (LC) cells (lanes 1-7) and ONH astrocytes (ONA) (lanes 8-11).L=base pair markers. C=PCR negative control lane. β-actin was used as apositive RT-PCR internal control.

FIG. 15. Illustrates increased expression of the BMP antagonist gremlin(CKTSF1B1) in glaucomatous TM cells. Gene expression was assessed usingAffymetrix gene arrays (Affymetrix gene chip U133A).

DETAILED DESCRIPTION PREFERRED EMBODIMENTS

The trabecular meshwork has been proposed to play an important role inthe normal flow of the aqueous humor, and has been presumed to be themajor site of outflow resistance in glaucomatous eyes. Human trabecularmeshwork (HTM) cells are specialized cells which line the outflowchannels by which aqueous humor exits the eye. Altered syntheticfunction of the cells may be involved in the pathogenesis of POAG,steroid glaucoma, and other types of glaucoma.

Despite years of intensive research, the precise molecular mechanismsresponsible for glaucomatous damage to the eye are not known. Recentresearch has suggested that growth factors may be important inmaintaining normal homeostasis in the ocular tissues associated withglaucoma, and alterations in growth factor/growth factor receptors mayplay a role in glaucoma pathogenesis. Growth factors area very largefamily of polypeptides that control cell growth and differentiation.These molecules have a variety of cell-specific effects on geneexpression, extracellular matrix composition and deposition,cytoskeletal organization, and regulation of cellular functions. The TMexpresses a wide variety of growth factors, growth factor receptors(Tripathi et al. 1993a; Tripathi et al. 1993b; Tripathi et al. 1994a;Tripathi et al. 1994b; Wordinger et al. 1998; Wordinger et al. 1999) aswell as neurotrophin/neurotrophic factors and their receptors (Liu etal. 2001; Wordinger et al. 2000). ONH astrocytes and lamina cribrosacells, two cell types of the optic nerve head, express growth factors,neurotrophins and their receptors (Lambert et al. 2001; Pena et al.1999). The aqueous humor also contains a variety of growth factorsincluding FGF2, EGF, TGFβ, HGF (Tripathi et al. 1996; Tripathi et al.1991; Tripathi et al. 1992; Hu and Ritch 2001) as well as neurotrophins(Chundru et al. 2000). Elevated levels of aqueous humor TGFβ-2 and HGFhave been reported in POAG patients (Tripathi et al. 1994c; Inatani etal. 2001; Picht et al. 2001). Growth factors may be involved in glaucomaby altering the normal development and/or function of the TM and ONH.

The present invention stems in part from the recognition that bonemorphogenic proteins (BMPs) not only induce bone and cartilage formationbut are multifunctional cytokines having a wide range of effects onnumerous cell types (Hogan 1996; Reddi 1997) and are expressed by bothhuman trabecular meshwork (HTM) and optic nerve head (ONH) cells(Wordinger et al. 2002). BMPs are members of the TGFβ superfamily, andthere are approximately 15-20 BMPs genes in man, 3 BMP receptors, and anumber of BMP associated proteins that function as BMP antagonists(Yamashita et al. 1996). BMPs signal via a receptor complex consistingof BMPR-I and BMPR-II. It has been reported that superfamily membersTGFβ and TGFβR (Agarwal et al. 1997; Lambert et al. 1997) and GDNF andGDNFR (Wordinger et al. 1999; Liu et al. 1999) are expressed by both HTMand ONH cells.

BMPs and BMP receptors are expressed in ocular tissues (Obata et al.1999; You et al. 1999), but previous reports have focused on oculardevelopment. The function of BMPs is important in ocular developmentsince targeted disruption of genes encoding BMPs in mice leads to severedevelopmental defects in the retina and the lens (Jena et al. 1997; Luoet al. 1995; Dudley et al. 1995). BMP-2, BMP-4 and BMP-7 are involved inthe development of the lens and retina (Jena et al. 1997; Furuta andHogan 1998; Reddi 2000; Trousse et al. 2001). BMP-6 and BMP-7 alsoappear to play a role in protecting neurons from hypoglycemic orischemic damage (Nonner et al. 2001; Liu et al. 2001), and BMP2 has beenshown to enhance ganglion cell neurotrophin expression (Zhang et al.1998). Heterozygous knock-out mice haploinsufficient for Bmp4 haveocular phenotypes including anterior segment dysgenesis, elevated IOP,and optic nerve abnormalities (Chang et al. 2001). There has been verylimited information published concerning the role of BMPs in the humanpostnatal eye.

Mohan and colleagues (1998) reported that BMP-2 and BMP-4 and BMPreceptors were expressed in cells of the adult cornea and suggested thatBMP function might include corneal keratocyte proliferation andapoptosis. You and colleagues (1999) verified this study and alsoreported the expression of BMP-3, BMP-5, and BMP-7 in ex vivo andcultured corneal epithelium and stromal cells. They reported that thelevel of BMP transcription was higher in the stroma while the level forreceptors was higher in cultured corneal epithelial cells.

Using RT-PCR, the present inventors discovered mRNAs for BMPs, BMPreceptors BMPR-IA, BMPR-IB and BMPR-II, as well as BMP binding proteinsgremlin, chordin, follistatin, and bambi, in HTM, lamina cribosa (LC)and ONH astrocyte cell lines and tissues (Wordinger et al. 2002). Thepresent inventors further discovered that HTM and ONH cells expressproteins BMP-2, BMP-4, BMP-5 and BMP-7.

Glaucoma will be diagnosed by characterization of genetic changes ingenes of members of the BMP signaling family. As used herein, thephrases “bone morphogenic protein family member gene” and “BMP signalingfamily” refer to all BMPs, BMP receptors and associated proteins. Theterm “genetic changes” is well known by those skilled in the art. Thereare numerous examples of diseases associated with genetic changes inspecific genes (for examples see Cummings 1997; Strachan, et al. 1996;Jorde, et al. 1999). Genetic changes in a specific gene (e.g. BMP) canbe determined using a variety of techniques well known by those skilledin the art, such as: SSCP, DGGE, ASO, RFLP, heteroduplex analysis, CCM,PTT, and RNase cleavage (see Birren, et al. 1998).

Glaucoma may be caused by altered expression of one or more BMP familygenes in the eye, which leads to elevated IOP and/or glaucomatous opticneuropathy. “Altered BMP gene expression” means expression of this geneproduct that is different from normal. The term may also refer toalterations in the sequence of the gene or protein. The normal BMP genehas been well characterized (see above), and the expression of BMP hasbeen reported in a variety of tissues including the TM and ONH. Geneticchanges in the coding region of BMP family genes may alter the functionof these proteins. Genetic changes outside the coding region may alsolead to glaucoma.

It is well known by those skilled in the art that “changes outside” ofthe coding region of a specific gene are important in the regulation ofgene expression. For example, the region upstream (5′) of the codingregion of most genes is known as the promoter region which “promotes”and regulates the expression of that gene. The promoter region containsnumerous nucleotide sequences recognized by various transcriptionfactors and DNA binding proteins that are responsible for activation orrepression of gene expression. Regions downstream (3′) of the gene candetermine poly-adenylation of the gene product, thereby regulating RNAprocessing and translation of the gene product.

The altered expression of BMP genes or mutations in the sequence of thegenes that is indicative of glaucoma may be detected using techniqueswell known to those of skill in the art. For example, it is contemplatedthat a nucleic acid fragment of almost any length may be employed, withthe total length preferably being limited by the ease of preparation anduse in the intended protocol. The nucleic acid sequences disclosedherein may also have utility as probes or primers in nucleic acidhybridization embodiments. As such, it is contemplated that nucleic acidsegments that comprise a sequence region that consists of at least a 14nucleotide long contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous sequence of BMP-2A(SEQ ID NO:1), BMP-4 (SEQ ID NO:3), BMP-5 (SEQ ID NO:5), BMP-7 (SEQ IDNO:7), BMP-RIA (SEQ ID NO:37), BMP-RIB (SEQ ID NO:39), BMP-RII (SEQ IDNO:41), chordin (SEQ ID NO:43), gremlin (SEQ ID NO:45), follistatin (SEQID NO:47), or bambi (SEQ ID NO:53) will find particular utility. Longercontiguous identical or complementary sequences, e.g., those of about20, 30, 40, 50, 100, 200, 500, 1000 nucleotides (including allintermediate lengths), and even up to full length sequences of about1547 nucleotides (for BMP-2A), 1946 nucleotides (for BMP-4), 2153nucleotides (for BMP-5) and 1878 nucleotides (for BMP-7), 2932nucleotides (for BMP-RIA), 2032 nucleotides (for BMP-RIB), 3611nucleotides (for BMP-RII), 3561 nucleotides (for chordin), 4049nucleotides (for gremlin), 1386 nucleotides (for follistatin), and 1523nucleotides (for bambi) will also be of use in certain embodiments.

It will be readily understood that “intermediate lengths”, in thiscontext, means any length between the quoted ranges, such as 14, 15, 16,17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52,53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; includingall integers through the 200-500; 500-1,000; 1,000-2,000 ranges, up toand including sequences of 2,001, 2002, 2050, 2051, and the like.

The ability of such nucleic acid probes and primers to specificallyhybridize to BMP coding sequences and primers to specifically amplifyBMP sequences will enable them to be of use in detecting the presence ofcomplementary sequences in a given sample. However, other uses areenvisioned, including the use of the sequence information for thepreparation of mutant species primers, or primers for use in preparingother genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of 10, 20, 30, 50, or even of 100-200 nucleotidesor so, identical or complementary to BMP-2A (SEQ ID NO:1), BMP4 (SEQ IDNO:3), BMP-5 (SEQ ID NO:5), BMP7 (SEQ ID NO:7), BMP-RIA (SEQ ID NO:37),BMP-RIB (SEQ ID NO:39), BMP-RII (SEQ ID NO:41), chordin (SEQ ID NO:43),gremlin (SEQ ID NO:45), follistatin (SEQ ID NO:47), or bambi (SEQ IDNO:53) are particularly contemplated as hybridization probes for use in,e.g., SNP evaluation and solid phase hybridization assays, in additionto Southern and northern blotting. This would allow BMP structural orregulatory genes to be analyzed, both in tissues and cells. The totalsize of fragment, as well as the size of the complementary stretch(es),will ultimately depend on the intended use of application of theparticular nucleic acid segment. Smaller fragments will generally finduse in hybridization embodiments, wherein the length of the contiguouscomplementary region may be varied, such as between about 10 and about100 nucleotides, but larger contiguous complementary stretches of up toabout 1547 nucleotides (for BMP-2A), 1946 nucleotides (for BMP-4), 2153nucleotides (for BMP-5) and 1878 nucleotides (for BMP-7), 2932nucleotides (for BMP-RIA), 2032 nucleotides (for BMP-RIB), 3611nucleotides (for BMP-RII), 3561 nucleotides (for chordin), 4049nucleotides (for gremlin), 1386 nucleotides (for follistatin), and 1523nucleotides (for bambi) may be used, according to the lengthcomplementary sequences one wishes to detect.

The use of a hybridization probe of about 10-14 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 10 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained, one will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 20 contiguous nucleotides,or even longer where desired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review thesequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47 or SEQ ID NO:53 and to select any continuous portionof the sequence, from about 10 nucleotides in length up to and includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences may be governed byvarious factors, such as, by way of example only, one may wish to employprimers from towards the termini of the total sequence, or from the endsof the functional domain-encoding sequences, in order to amplify furtherDNA.

The process of selecting, and preparing a nucleic acid segment thatincludes a contiguous sequence from within BMP-2A (SEQ ID NO:1), BMP4(SEQ ID NO:3), BMP-5 (SEQ ID NO:5), BMP7 (SEQ ID NO:7), BMP-RIA (SEQ IDNO:37), BMP-RIB (SEQ ID NO:39), BMP-RII (SEQ ID NO:41), chordin (SEQ IDNO:43), gremlin (SEQ ID NO:45), follistatin (SEQ ID NO:47), or bambi(SEQ ID NO:53) may alternatively be described as preparing a nucleicacid fragment. Of course, fragments may also be obtained by othertechniques such as, e.g., by mechanical shearing or by restrictionenzyme digestion. Small nucleic acid segments or fragments may bereadily prepared by, for example, directly synthesizing the fragment bychemical means, as is commonly practiced using an automatedoligonucleotide synthesizer. Also, fragments may be obtained byapplication of nucleic acid reproduction technology, such as the PCR™technology of U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195 (eachincorporated herein by reference), by introducing selected sequencesinto recombinant vectors for recombinant production, and by otherrecombinant DNA techniques generally known to those of skill in the artof molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of BMP genes or cDNAs. Depending on the applicationenvisioned, one will desire to employ varying degrees of selectivity ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by 0.02M-0.15M NaCl attemperatures of 50° C. to 70° C. Such selective conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand, and would be particularly suitable for examining BMP genes.

Of course, for some applications, for example, where one desires toprepare or identify mutants employing a mutant primer strand hybridizedto an underlying template or where one seeks to isolate BMP encodingsequences from related species, functional equivalents, or the like,less stringent hybridization conditions will typically be needed inorder to allow formation of the heteroduplex. In these circumstances,one may desire to employ conditions such a 0.15M-1.0M salt, attemperatures ranging from 20° C. to 55° C. Cross-hybridizing species canthereby be readily identified as positively hybridizing signals withrespect to control hybridizations. In any case, it is generallyappreciated that conditions can be rendered more stringent by decreasingNaCl concentrations or by the addition of increasing amounts offormamide, which serves to destabilize the hybrid duplex in the samemanner as increased temperature. Thus, hybridization conditions can bereadily manipulated, and thus will generally be a method of choicedepending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C contents, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

It will also be understood that this invention is not limited to theparticular nucleic acid and amino acid sequences of BMP-2A (SEQ IDNO:1), BMP4 (SEQ ID NO:3), BMP-5 (SEQ ID NO:5), BMP7 (SEQ ID NO:7),BMP-RIA (SEQ ID NO:37), BMP-RIB (SEQ ID NO:39), BMP-RII (SEQ ID NO:41),chordin (SEQ ID NO:43), gremlin (SEQ ID NO:45), follistatin (SEQ IDNO:47), or bambi (SEQ ID NO:53). Recombinant vectors and isolated DNAsegments may therefore variously include the BMP coding regionsthemselves, upstream or downstream regions of the genes, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides that nevertheless includeBMP coding regions or may encode biologically functional equivalentproteins or polypeptides that have variant amino acid sequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent BMP proteins and polypeptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencythat are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor polypeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test BMP mutantsin order to examine binding activity at the molecular level.

The therapeutic agent for the treatment of glaucoma can be: a peptide orprotein, a peptide mimetic, an oligonucleotide or derivatizedoligonucleotide, or small drug-like molecule, all which affect one ormore aspects of the ocular BMP pathways. Preferred therapeutic agentsare those that are: (1) BMP2, BMP4, BMP5, or BMP7 agonists; (2) chordin,gremlin, follistatin, or bambi antagonists; and/or (3) Smad1, Smad5and/or Smad4 agonists.

The agent may be delivered directly to the eye (for example: topicalocular drops or ointments; slow release devices in the cul-de-sac orimplanted adjacent to the sclera or within the eye; periocular,conjunctival, sub-Tenons, intracameral or intravitreal injections) orparenterally (for example: orally; intravenous, subcutaneous orintramuscular injections; dermal delivery; etc.) using techniques wellknown by those skilled in the art. The following are examples ofpossible formulations embodied by this invention.

wt. % (a) Topical ocular formulation Agent that increases ocular BMP-4expression 0.01-2 HPMC 0.5 Sodium chloride 0.8 BAC 0.01% EDTA 0.01NaOH/HCl qs pH 7.4 Purified water qs 100 mL (b) Topical ocularformulation Gremlin antagonist 0.01-2 HPMC 0.5 Sodium chloride 0.8 BAC0.01 EDTA 0.01 NaOH/HCl qs pH 7.4 Purified water qs 100 mL (c) Topicalocular formulation Smad 1/5 agonist 0.01-2 HPMC 0.5 Sodium chloride 0.8BAC 0.01 EDTA 0.01 NaOH/HCl qs pH 7.2 Purified water qs 100 mL

It is further contemplated that the compounds of the invention could beformulated in intraocular insert devices.

A. Assay for Therapeutic Agents

This invention is also useful for the discovery of new anti-glaucomatherapeutic agents that are involved in the BMP signaling pathway (seeFIG. 5). Selective BMP ligands bind to BMP type I and type IIserine/threonine kinase receptors (BMP-RI and BMP-RII) and transducesignal via Smad proteins. The BMP signal is propagated by Smads throughprotein-protein and protein-DNA interactions (Attisano and TuenLee-Hoeflich 2001). Regulatory Smad 1 and Smad 5 are activated (viaphosphorylation) by ligand bound BMP receptors (von Bubnoff and Cho2001). These regulatory Smads then interact with Smad 4 to form aheteromeric complex that translocates to the nucleus. This complex isable to activate or repress the transcription of selective genes thatrecognize this transcriptional complex, depending on which nuclearco-factors are present.

The BMP/Smad signaling pathway is negatively regulated by severalmechanisms. Certain BMP-binding proteins (such as gremlin, BAMBI, orfollistatin) bind BMPs and inhibit their interaction with BMP receptors.In addition, there are inhibitory Smad proteins (e.g. Smad 6 and Smad7), which bind and inactivate BMP receptors. (Kowabata et al. 1998; Itohet al. 2000; Miyazono 2000). The present inventors have discovered thathuman TM cells, ONH astrocytes and lamina cribrosa cells express messageand protein for the BMP receptor complex. Thus, these cells couldrespond to endogenous BMP ligands.

Various methods may be used to discover new anti-glaucoma therapeuticagents, and these techniques are well known to those skilled in the art.For example, peptide or peptide mimetic agents that act as agonists orinhibitors of BMPs can be discovered through molecular modeling ofBMP/BMP receptor structures (Nickel et al. 2001). BMP signaltransduction involves select sets of Smad proteins (Kawabata et al.1998; Itoh et al. 2000; Attiseno et al. 2000). Select BMP agonists andSmad agonists can be discovered using cell based assays. The test cellshould express the appropriate BMP receptor(s) and possess theappropriate BMP signaling pathway. Because one of the major effects ofBMP signaling is the alteration of gene expression, BMP agonists andSmad agonists can be discovered by screening for BMP-induced genes. Theinduction of BMP regulated genes also may be assayed by quantitatinglevels of mRNA using quantitative RT-PCR (Wang et al. 2001), DNAmicroarrays, or reporter gene constructs. There are natural inhibitorsof BMP signaling, the BMP binding proteins (also known as BMP-associatedproteins), such as chordin, gremlin, and follistatin. Antagonists of theprotein inhibitors can be discovered using ligand binding assays. Forexample, test agents can be added to recombinant purified gremlin, andthose agents that bind to gremlin are identified using a variety oftechniques known to those skilled in the art. To determine whether theseagents are gremlin antagonists, a cell based assay similar to thatdescribed above is used.

It is contemplated that any known in vitro and in vivo screening modelsmay be used in conjunction with the present invention to identify newglaucoma therapies directed to the BMP family of genes. Such models arewell known to those skilled in the art and their practice has becomeroutine. Small peptides or peptide mimetics can be designed based onstructure/function knowledge of the BMP, BMPR, and/or BMP bindingprotein gene products. Ligand binding assays can be used to detect smallmolecules that bind to BMPs, BMPRs, or BMP binding proteins. Cell basedassays can look at the effects of various agents on BMP signalingpathways. Knock-in cell lines containing BMP family gene promoterscoupled to a reporter gene can be generated to look for agents thatalter BMP family member gene expression. These assays can be used toidentify both agonist and antagonist molecules. Ex vivo assays, such asperfusion cultured anterior segments from human eyes (Clark et al.1995a; Pang et al. 2000), can be used to examine the effects of agentson IOP and on BMP signaling in TM tissue. Rodent models of glaucoma canbe generated using well-known techniques to create stable BMP familymember transgenic, knockout, or knock-in strains of mice and rats. Theserodent models can be used to screen for agents that alter theglaucoma-like phenotype(s) (e.g. tonometry to evaluate effects on IOP,histology to evaluate effects on glaucomatous optic neurology).

B. Kits

The present invention provides methods, compositions and kits for theearly detection of glaucoma. The kits can contain a nucleic acid segmentencoding a BMP polypeptide or protein. The kit can further containreagents for detecting an interaction between a sample and a nucleicacid or peptide of the present invention. The provided reagent can beradio-, fluorescently- or enzymatically-labeled. The kit can contain aknown radiolabeled agent capable of binding or interacting with anucleic acid or peptide or protein of the present invention.

The reagent of the kit can be provided as a liquid solution, attached toa solid support or as a dried powder. Preferably, when the reagent isprovided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent provided is attached to a solidsupport, the solid support can be chromatography media, a test platehaving a plurality of wells, or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concerns diagnosticmethods and associated kits for the diagnosis of glaucoma. It isproposed that the BMP associated peptides and nucleic acids of theinvention may be employed to detect polymorphisms or mutations in theBMP nucleic acids from patient samples. In general, these methods willinclude first obtaining a sample suspected of containing such apolymorphism or mutation, contacting the sample with a peptide ornucleic acid of the present invention, as the case may be, underconditions effective to allow the formation of a complex, and thendetecting the presence of the complex.

In general, the detection of complex formation is quite well known inthe art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, indirect fluorescence techniques and thelike. Generally, complex formation will be detected through the use of alabel, such as a radiolabel or an enzyme tag (such as alkalinephosphatase, horseradish peroxidase, or the like). Of course, one mayfind additional advantages through the use of a secondary bindingligand.

The following examples are representative of the techniques employed bythe inventors in carrying out aspects of the present invention. Itshould be appreciated that while these techniques are exemplary ofpreferred embodiments for the practice of the invention, those of skillin the art, in light of the present disclosure, will recognize thatnumerous modifications can be made without departing from the spirit andintended scope of the invention.

Example 1

Cell culture: Human TM cells and ONH cells were generated from donoreyes as described (Steely et al. 1992; Steely et al. 2000; Wilson et al.1993; Clark et al. 1994; Clark et al. 1995b; Clark et al. 1995c; Clarket al. 1996; Clark et al. 2001a; Clark et al. 2001b; Dickerson et al.1998; Wordinger et al. 1998; Wordinger et al. 1999; Wordinger et al.2000; Wordinger et al. 2002; Lambert et al. 2001; Agarwal et al. 1999;Liu et al. 2001). TM cells were grown from TM explants of donors rangingin age from 6 days to 90 years. Human optic nerve head astrocytes andlamina cribrosa (LC) cells were generated from carefully dissected opticnerve heads (donors aged 2 days to 90 years) and characterized accordingto previous reports (Lambert et al. 2001; Clark et al. 1995a). The cellswere grown to confluency in the following media: Ham's F10 media (JRHBiosciences, Lenexa, Kans.) containing 10% fetal bovine serum (HyClone,Logan, Utah) and antibiotics (Gibco BRL-Life Technologies, Grand Island,N.Y.) for TM cells; Dulbecco's modified Eagle's media (DMEM, HyClone)containing 10% FBS for LC cells; and astrocyte growth medium (AGM,Clonetics, San Diego, Calif.) containing 5% FBS for ONH astrocytes.

RT-PCR: Human TM and ONH tissues also were dissected from donor eyes(Wordinger et al. 1998; Wang et al. 2001). Total RNA was extracted fromthe TM and ONH cells and tissues using TRIzol extraction (Gibco BRL-LifeTechnologies), and cDNA prepared by reverse transcription using standardprocedures (Wordinger et al. 1998; Wordinger et al. 1999; Wordinger etal. 2000; Wordinger et al. 2002). PCR primers were designed using theOligos 4.0 software program (see primer pairs in Table 1). All primerpairs were designed so that amplification of potentially contaminatedgenomic DNA sequences would produce mRNA PCR products that would besubstantially larger than expected, because intron sequences that wereexcised during RNA processing would be included in genomic DNA. Theβ-actin PCR primers, AGGCCAACCGCGAGAAGATGACC (upstream) (SEQ ID NO: 55)and GAAGTCCAGGGCGACGTAGCAC (downstream) (SEQ ID NO: 56) with anannealing temperature of 55° C. yielded a PCR product of 350 bp.

PCR reactions were run as described (Wordinger et al. 1998; Wordinger etal. 1999; Wordinger et al. 2000; Lambert et al. 2001; Wordinger et al.2002) using Taq Start Antibody Hot Start with the following cycleconditions: 2 minutes at 94° C., 2 minutes at 92° C., and 40 cycles of30 seconds at the optimal annealing temperature, extension for 90seconds at 72° C. and denaturation for 45 seconds at 92° C. Theamplified PCR products were examined by horizontal electrophoresis in1.5% agarose gels. To ensure specificity of the RT-PCR products,Southern blot analysis was performed with probes designed using Oligo4.0 that hybridized to a region within the amplified PCR product. PCRproducts were sequenced to verify the specificity of the PCR reactions.Table 2 lists the members of the BMP family that are expressed in thehuman TM and ONH.

TABLE 1 PCR Primer Pairs, Annealing Temperature and Amplimer Size ofBMPs Ampl. Assession Size Name Number Upstream PCR Primer Downstream PCRPrimer (bp) BMP-2A NM_001200 ACTGCGGTCTCCTAAA GCTGACCTGAGTGCCT 657GGTCGA (SEQ ID NO: 9) GCGAT (SEQ ID NO: 10) BMP-4 NM_001202GAATGCTGATGGTCGT AGACTGAAGCCGGTA 348 TTTTATTATG (SEQ ID AAGAT (SEQ IDNO: 12) NO: 11) BMP-5 NM_021073 AAGAGGACAAGAAGG GTAGAGATCCAGCATA 303ACTAAAAATAT (SEQ AAGAGAGGT (SEQ ID ID NO: 13) NO: 14) BMP-7 NM_001719AGCCCGGGTAGCGCGT GCGCCGGTGGATGAA 202 AGAG (SEQ ID NO: 15) GCTCGA (SEQ IDNO: 16) BMPR-1A NM_004329 TAAAGGTGACAGTACA TCTATGATGGCAAAGC 298 CAGGAACA(SEQ ID AATGTCC (SEQ ID NO: 17) NO: 18) BMPR-1B NM_001203TACAAGCCTGCCATAA ATCATCGTGAAACAAT 211 GTGAAGAAGC (SEQ ID ATCCGTCTG (SEQID NO: 19) NO: 20) BMPR-II NM_001204 TCCTCTCATCAGCCAT AGTTACTACACATTCT457 TTGTCCTTTC (SEQ ID TCATAG (SEQ ID NO: 21) NO: 22) Chordin AF209930CTCTGCTCACTCTGCA CCGGTCACCATCAAAA 198 (CHRD) CCTG (SEQ ID NO: 23) TAGC(SEQ ID NO: 24) Gremlin NM_013372 ATCAACCGCTTCTGTT ATGCAACGACACTGCT 197(CKTSF1 ACGG (SEQ ID NO: 25) TCAC (SEQ ID NO: 26) B1) FollistatinNM_006350 TGCCACCTGAGAAAGG ACAGACAGGCTCATCC 201 (FST) CTAC (SEQ ID NO:27) GACT (SEQ ID NO: 28) Noggin NM_005450 CACTACGACCCAGGCTCTCCGCAGCTTCTTGC 212 (NOG) TCAT (SEQ ID NO: 29) TTAG (SEQ ID NO: 30)CER-1 NM_005454 ATAGTGAGCCCTTCCC AATGAACAGACCCGC 294 ACCT (SEQ ID NO:33) ATTTC (SEQ ID NO: 34) NMA NM_005791 GATCGCCACTCCAGCTGGGCACGGCAATGACC 471 (BAMBI) ACATC (SEQ ID NO: 35) (SEQ ID NO: 36)

TABLE 2 BMP Family Members Expressed in Human TM and ONH TrabecularOptic Nerve BMP Family Member Meshwork Head BMP-2 + + BMP-4 + +BMP-5 + + BMP-7 + + BMPR-IA + + BMPR-IB + + BMPR-II + + Chordin + +Gremlin + + Follistatin + + Bambi + + Noggin − − CER-1 − −

Western immunoblotting: Protein was extracted from cultured cells usinglysis buffer, and proteins were separated by denaturing polyacrylamidegel electrophoresis prior to electrophoretic transfer to nitrocellulosemembranes (Lambert et al. 2001). The membranes were blocked with 5% milk(for BMPs) or 3% gelatin (for BMPRs) and incubated with the followingprimary antibodies: BMP2, BMP4, BMP5, BMP7 (all from Santa Cruz, SantaCruz, Calif.), or BMP-RIA, BMP-RIB, BMP-RII (from Jackson ImmunoResearch, West Grove, Pa.). The membranes were washed, incubated withsecondary antibodies (goat anti-mouse IgG-horseradish peroxidase forBMPs, Santa Cruz; donkey anti-goat-horseradish peroxidase for BMPreceptors, Jackson Immuno Research), and developed using theWesternBreeze chemiluminescence immunodetection system (Invitrogen,Carlsbad, Calif.).

Expression of BMPs, BMPRs mRNA in human TM cells and tissues:Amplification products of expected for BMP-2, BMP-4, BMP-5 and BMP-7primer pairs in human TM cells and tissues are shown in FIG. 6. Southernblots using specific probes verified that these were the expected PCRproducts. All human TM cell lines and tissues expressed message forBMP-2, BMP-4, and BMP-7. However, message for BMP-5 was low toundetectable in human TM tissue samples (FIG. 6, lanes 6 and 7). Controlreactions without cDNA did not result in amplification productsindicating that reagents and primers were free of DNA or RNAcontamination (FIG. 6, lane C).

FIG. 7 shows the amplification products of expected size for BMP-RIA,BMP-RIB, and BMP-RII primer pairs in human TM cells and tissues. Allhuman TM cells and tissues expressed message for the BMP receptorcomplexes. Southern blots using specific probes verified that these werethe expected PCR products. An alternate amplification product (350 bp)was detected in the BMP-RII PCR reaction. The alternate amplificationproduct was present in all human TM cells and tissues. This alternateband is currently being identified to determine if it is an alternatespliced form of the receptor. Control reactions without cDNA did notresult in amplification products (FIG. 7, lane C) indicating thatreagents and primers were free of DNA or RNA contamination.

Expression of BMP and BMP receptor mRNA in human ONH cells and tissues:Amplification products of expected size for BMP-2, BMP-4, BMP-5 andBMP-7 primer pairs in human ONH astrocytes and ONH tissues are shown inFIG. 8. All ONH astrocytes and ONH tissue expressed message for therespective BMP. Human brain astrocytes were used as a positive controlcell line. Southern blots using specific probes verified that these werethe expected PCR products. With the exception of BMP-2, all other BMPwere expressed by human brain astrocytes (FIG. 8, lane 7). Controlreactions without cDNA did not result in amplification products (FIG. 8,lane C) indicating that reagents and primers were free of DNA or RNAcontamination.

FIG. 9 shows the amplification products of expected sizes for BMP-2,BMP-4, BMP-5 and BMP-7 primer pairs in cultured human LC cells. All LCcell lines expressed message for each BMP. Southern blots using specificprobes verified that these were the expected PCR products. Controlreactions without cDNA did not result in amplification products (FIG. 9,lane C) indicating that reagents and primers were free of DNA or RNAcontamination.

Amplification products of expected size for BMP-RIA, BMP-RIB, andBMP-RII primer pairs in human ONH astrocytes and ONH tissues are shownin FIG. 10. All ONH astrocyte cell lines and tissues expressed messagefor BMP-RIA and BMP-RIB. Southern blots using specific probes verifiedthat these were the expected PCR products. With the exception of ONHtissue (FIG. 10, lane 6), BMP-RII was expressed by all ONH astrocytecell lines. Message for all BMP receptors (FIG. 10, lane 7) wasexpressed by a human brain astrocyte cell line that served as a positivecontrol. There appears to be a discrepancy in the expression of BMP-RIIin ONH tissue and ONH cell lines. The reduced expression in ONH tissuemay reflect a low level of expression. Control reactions without cDNAdid not result in amplification products (FIG. 5, lane C) indicatingthat reagents and primers were free of DNA or RNA contamination.

FIG. 11 shows the amplification products of expected size for BMP-RIA,BMP-RIB, and BMP-RII primer pairs in cultured human LC cells. All LCcell lines expressed message for each BMP receptor. Southern blots usingspecific probes verified that these were the expected PCR products.Control reactions without cDNA did not result in amplification products(FIG. 11, lane C) indicating that reagents and primers were free of DNAor RNA contamination.

Expression of BMP proteins and BMP receptor proteins in human TM and ONHcells and tissues: FIG. 12 represents chemiluminescent immunoblotdetection of BMP-2, BMP-4, BMP-5, BMP-7, BMP-RIA, BMP-RIB, and BMP-RIIproteins in human TM and ONH cells and tissues. All cell lines studiedexpressed the respective BMP proteins. The BMP proteins were detected incell lines the following molecular weights: 54-56 kDa for BMP-2, 25-27kDa for BMP-4, 55-57 kDa for BMP-5, and 77 kDa for BMP-7. Multiple bandswere detected for BMP-2 and BMP-4, which most likely representglycosylated, and partially glycosylated forms of these BMPs as seen inother studies. However, we did not do glycosylation studies as they werebeyond the scope of this study. The BMP receptor proteins were detectedin cell lines at molecular weights: 38 kDa for BMP-RIA, 64 kDa forBMP-RIB, and 57 kDa for BMP-RII. Multiple bands were detected forBMP-RIB and BMP-RII in the TM cells, which most likely representglycosylated, and partially glycosylated forms as seen in other studies.The expression levels of proteins for the BMP receptors appeared to belower in the TM cells compared to ONH cells. For example BMP-RII was notdetected in TM cells and BMP-RIB was greatly reduced.

Expression of BMP associated protein mRNAs in cultured human TM cellsand in human ONH cells: Amplification products of expected size for BMPassociated protein primer pairs in human TM cell lines are shown in FIG.13. Human TM cell lines expressed message for DRM (gremlin), chordin,follistatin, and NMA (BAMBI). Southern blots using specific probesverified that these were the expected PCR products. There was noapparent difference in message expression between cell lines. All humanTM cells examined failed to express mRNA for the BMP associated proteinsnoggin and Cer-1. Control reactions without cDNA did not result inamplification products indicating that reagents and primers were free ofDNA or RNA contamination.

Amplification products of expected size for BMP associated proteinprimer pairs in ONH astrocytes and LC cell lines are shown in FIG. 14.All ONH astrocytes and LC cell lines expressed message for DRM(gremlin), follistatin and NMA (BAMBI). Southern blots using specificprobes verified that these were the expected PCR products. The majorityof LC cells and ONH astrocytes expressed message for chordin. All humanONH astrocytes and LC cells examined failed to express mRNA for the BMPassociated proteins noggin and Cer-1. Control reactions without cDNA didnot result in amplification products indicating that reagents andprimers were free of DNA and RNA contamination.

FIG. 15 shows increased expression of the BMP antagonist gremlin(CKTSF1B1) in glaucomatous TM cells. Gene expression was assessed usingAffymetrix gene arrays (Affymetrix gene chip U133A).

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically and structurallyrelated may be substituted for the agents described herein to achievesimilar results. All such substitutions and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for treating glaucoma comprising administering to a patientin need thereof a composition comprising human bone morphogenic protein7 (hBMP7).